Field of the Invention
The invention relates to the field of molecular biology. It particularly relates to methods to improve the efficiency of directed Integration of nucleic acids into the genome of a filamentous fungus and uses thereof.
Description of Related Art
Eukaryotic cells are preferred organisms fur (recombinant) production of polypeptides and secondary metabolites. When constructing, for example, a protein production strain, the site of integration of the gene of interest coding for the protein to be produced is crucial for the regulation of transcription and/or expression of the integrated gene of interest. Since in most eukaryotic organisms integration of DNA into the genome occurs with high frequency at random, the construction of a protein production strain by recombinant DNA technology often leads to the unwanted random integration of the expression cassette comprising the gene encoding the protein to be produced. This uncontrolled “at random multiple integration” of an expression cassette is a potentially dangerous process, which can lead to unwanted modification of the genome of the host. It is therefore highly desirable to be able to construct a protein production strain by ensuring the correct targeting of the expression cassette with high efficiency. Furthermore, now that the sequence of complete genomes of an increasing amount of organisms is becoming available, this opens the opportunity to construct genome spanning overexpression and deletion libraries. An important requirement for the efficient construction of such libraries is that the organism. In question can be efficiently transformed and that the required homology needed to direct targeted integration of a nucleic acid into the genomes relatively short.
Eukaryotic cells have at least two separate pathways (one via homologous and one via non-homologous recombination) through which nucleic acids (in particular of course DNA) can be integrated into the host genome. The yeast Saccharomyces cerevlsiaa is an organism with a preference for homologous recombination (HR). The ratio of non-homologous to homologous recombination (NHR/HR) of this organism may vary from about 0.07 to 0.007.
WO 02/052026 discloses mutants of Saccharomyces cerevisiae having an improved targeting efficiency of DNA sequences into its genome. Such mutant strains are deficient in a gene involved in NHR (KU70).
Contrary to Saccharomyces cerevisiae, most higher eukaryotes such as filamentous fungal cells up to mammalian cell have a preference for NHR. Among filamentous fungi, the NHR/HR ratio is ranged between 1 and more than 100. In such organisms, targeted integration frequency is rather low. To improve this frequency, the length of homologous regions flanking a polynucleotide sequence to be integrated into the genome of such organisms has to be relatively long for example at least 2000 bp for disrupting a single gene and at least 500 bp for screening putative transformants. The necessity of such flanking regions represents a heavy burden when cloning the DNA construct comprising said polynucleotide and when transforming the organism with it. Moreover, neighbouring genes which IIe within those flanking regions can easily be disturbed during the recombination processes following transformation, thereby causing unwanted and unexpected side-effects.
Mammalian cells deficient in KU70 have already been isolated (Pierce et al, Genes and Development, (2001), 15: 3237-3242). These mutants have a six-fold higher homology-directed repair frequency, but no increase in the efficiency of homology-directed targeted integration. This suggests that results obtained in organisms with a preference for HR (Saccharomyces cerevisiae) cannot be extrapolated to organisms with a preference for NHR.
Surprisingly, we found that steering the integration pathways of nucleic acids towards HR in filamentous fungi resulted in an improved efficiency for targeted integration of nucleic acids into the genome of filamentous fungi.